Activation of KRAS promotes the mesenchymal features of basal-type breast cancer

Abstract

Basal-type breast cancers are among the most aggressive and deadly breast cancer subtypes, displaying a high metastatic ability associated with mesenchymal features. However, the molecular mechanisms underlying the maintenance of mesenchymal phenotypes of basal-type breast cancer cells remain obscure. Here, we report that KRAS is a critical regulator for the maintenance of mesenchymal features in basal-type breast cancer cells. KRAS is preferentially activated in basal-type breast cancer cells as compared with luminal type. By loss and gain of KRAS, we found that KRAS is necessary and sufficient for the maintenance of mesenchymal phenotypes and metastatic ability through SLUG expression. Taken together, this study demonstrates that KRAS is a critical regulator for the metastatic behavior associated with mesenchymal features of breast cancer cells, implicating a novel therapeutic target for basal-type breast cancer.

Figure 1

KRAS activity is critical for mesenchymal phenotypes of basal-type breast cancer cells. (a) Activated KRAS affinity precipitation assay and western blot for KRAS comparing basal- and luminal-type of breast cancer cell lines. Migration and invasion assay of MDA-MB231 (b) and BT549 (c) basal-type breast cancer cells after treatment with siRNA targeting KRAS. β-Actin was used as a loading control. Error bars represent mean±s.d. of triplicate samples. KRAS, Kirsten rat sarcoma viral oncogene homolog; si-cont, scrambled control siRNA; si-KRAS, siRNA targeting KRAS. *P<0.01 versus control.

Figure 2

KRAS is necessary for the maintenance of mesenchymal features of basal-type breast cancer cells. Western blot analysis (a) and immunocytochemistry (b) for EMT markers such as FN1, VIM, E-cadherin and N-cadherin in basal-type breast cancer cells after treatment with siRNA targeting KRAS. (c) Western blot analysis for EMT master regulators such as SLUG, SNAIL, ZEB1 and TWIST in basal-type MDA-MB231 breast cancer cells after treatment with siRNA targeting KRAS. β-Actin was used as a loading control. DAPI, 4, 6-diamidino-2-phenylindole; KRAS, Kirsten rat sarcoma viral oncogene homolog; si-cont, scrambled control siRNA; si-KRAS, siRNA targeting KRAS; VIM, vimentin. Error bars represent mean±s.d. of triplicate samples. *P<0.01 versus control.

Figure 3

Exogenous expression of KRAS in luminal-type breast cancer cells. Western blot analysis for KRAS (a) and its major downstream effectors, RAF-1/ERK (b) and PI3K/AKT (c) in luminal-type MCF7 breast cancer cells that are transduced with KRAS or control vector MFG. β-Actin was used as a loading control. KRAS, Kirsten rat sarcoma viral oncogene homolog.

Figure 4

Exogenous expression of KRAS promotes migration and invasion of luminal-type breast cancer cells through EMT. Wound healing (a), migration and invasion assays (b) of MCF7 and SKBR3 luminal-type breast cancer cells that are transduced with KRAS or control vector MFG. Western blot analysis for EMT markers (c) and EMT regulators (d) in KRAS or control vector MFG-transduced MCF7 and SKBR3 luminal-type breast cancer cells. β-Actin was used as a loading control. Error bars represent mean±s.d. of triplicate samples. *P<0.01 versus control. KRAS, Kirsten rat sarcoma viral oncogene homolog; VIM, vimentin.

Figure 5

Exogenous expression of KRAS promotes migration and invasion of normal mammary cells through EMT. (a) Migration and invasion assays of MCF10A normal mammary cells that are transduced with KRAS or control vector MFG. Western blot analysis for EMT markers (b) and EMT regulators (c) in KRAS or control vector MFG-transduced MCF10A normal mammary cells. β-Actin was used as a loading control. Error bars represent mean±s.d. of triplicate samples. *P<0.01 versus control. KRAS, Kirsten rat sarcoma viral oncogene homolog; VIM, vimentin.

Figure 6

KRAS has a correlation with in vivo metastatic ability of basal-type breast cancer cells and prognosis of breast cancer patients. (a) Schematic experimental procedure for tail vein injection of breast cancer cells that are transfected with siRNA targeting KRAS or scrambled control siRNA into athymic BALB/c nude mice. (b) Representative images of whole-lung metastasis (left) and quantification of the metastases foci (right) generated by MDA-MB231 cells after tail vein injection (n=4). (c) Kaplan–Meier survival curves of human breast cancer patients using publicly available clinical breast cancer database. Breast cancer patients (3455) were grouped to high (2352) and low expression (1103) of KRAS. Note that KRAS expression level has a correlation with poor survival rate. KRAS, Kirsten rat sarcoma viral oncogene homolog. si-cont, scrambled control siRNA; si-KRAS, siRNA targeting KRAS. *P<0.01 versus control.